Surface-based biosensors are perhaps the most common type of sensors for biological targets such as nucleic acids and proteins. In most implementations, they are based on a “capturing probe” (e.g. an antibody or synthetic DNA sequence) which is immobilized on a surface, and to which targets specifically bind. Detection of the binding events can then be obtained in various ways, including for example fluorescence, electrochemical signals, or surface plasmon resonance (SPR).
Regardless of the binding or transduction mechanism, the sensitivity of all surface biosensors is fundamentally limited by the rate at which target molecules bind to the surface. Several factors, namely diffusion, transport, and reaction rates limit hybridization or binding at low concentrations. While diffusion and transport limitation can be effectively overcome by use of devices such as mixers and flow channels, reaction rates remain a major bottleneck toward achieving rapid binding of biomolecules at low concentrations. This is because hybridization and binding typically take the form of second order reactions, with reaction time inversely proportional to the concentration of the reactants (Squires, T. M., et al., 2008, Nat Biotechnol 26, 417-426). For example, surface-bound probes for nucleic acid capture are effectively employed in microarray technology. The main advantage of surface-bound probes are that they are easily multiplexed, thus enabling simultaneously screening for thousands or even up to 1 million biomarkers in a single experiment. However, the capture process is limited by both diffusion and slow reaction kinetics, and so incubation times typically exceed 24 hours. As a result, there is a growing need for methods that significantly accelerate reaction rates and lower detection time.
Isotachophoresis (“ITP”) is an electrophoresis technique which allows for simultaneous separation and preconcentration of analytes based on their effective electrophoretic mobility. The process has been described repeatedly, as for instance, Bier and Allgyer, Electrokinetic Separation Methods 443-69 (Elsevier/North-Holland 1979). As illustrated in FIG. 1, ITP uses a discontinuous buffer system consisting of leading (LE) and terminating (TE) electrolytes. The LE and TE are chosen to have respectively higher and lower electrophoretic mobility than the analytes of interest. Sample is injected between the TE and LE (or can be mixed with the TE in the reservoir). When an electric field is applied, ions whose electrophoretic mobility is bracketed between that of the LE and TE focus within an electric field gradient at the LE-TE interface. Design of the LE and TE chemistries enables selective focusing of species of interest, and exclusion of undesired species. Up to a million-fold increase in concentration in 2 minutes has been demonstrated.
ITP is typically used to focus a sample of interest and deliver a high concentration target to a pre-functionalized surface, thus enabling rapid reaction at the sensor site. A recent publication showed two orders of magnitude improvement in limit of detection (LoD) compared to standard continuous flow-based hybridization, in a 3 min ITP-based nucleic acid hybridization assay (Karsenty et al., 2014, Analytical chemistry, 86(6), 3028-3036. As shown in FIG. 2, the ITP interface in which the sample is focused, transverses by electromigration over the reactive surface. However, despite a 20,000-fold increase in sample concentration, signal is enhanced only 100-fold due to the short reaction time (2 sec) in which the sample overlaps with the surface. Allowing longer reaction times is key in exhausting the full potential of the technique.
US 2012/0175258 provides an isotachophoresis system for separating a sample containing particles into discrete packets including a flow channel having a large diameter section and a small diameter section; a negative electrode operably connected to the flow channel; a positive electrode operably connected to the flow channel; a leading carrier fluid in the flow channel; a trailing carrier fluid in the flow channel; and a control for separating the particles in the sample into discrete packets using the leading carrier fluid, the trailing carrier fluid, the large diameter section, and the small diameter section.
Thus, there is an unmet need for fully automated ITP devices and methods of use thereof, such as for performing assays having significantly accelerate reaction rates and lower detection time of analytes of interest.